Indian Journal of Allergy, Asthma and Immunology

: 2021  |  Volume : 35  |  Issue : 2  |  Page : 55--60

Qualitative analysis of protein antigen in bird exposed asthma patients by high-performance liquid chromatography method

Anil Kumar Mavi1, Sonam Spalgais1, Manoj Kumar1, Kamal Singh1, Priya2, Raj Kumar1,  
1 Department of Pulmonary Medicine, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
2 Department of Research, Mahavir Cancer Institute and Research Centre, Patna, Bihar, India

Correspondence Address:
Prof. Raj Kumar
Department of Pulmonary Medicine, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi - 110007


BACKGROUND: Asthma is a chronic disease that affects large number of people worldwide. In urban areas, asthmatic people are getting exposed to pigeon antigen (droppings and feathers) around the workplace and domestic areas. The present study was conducted to the determination of MUC1 protein in the blood of pigeon-exposed asthmatic patients by the high-performance liquid chromatography (HPLC) method. MATERIALS AND METHODS: This was a prospective study of 108 pigeon-exposed asthma patients. We enrolled 17 asthmatic patients for detection of MUC1 expression in their serum sample who were atopic and skin prick testing positive for pigeon antigens (droppings and feathers). The MUC1 analysis was done with HPLC method. RESULTS: A total of 17 asthmatic patients were enrolled on the basis of the history of pigeon exposure and positive skin prick test for pigeon's feather and dropping allergens including 9 males and 8 females, the average age of the study candidates was 28.8 ± 10.1 years. MUC1 concentration was raised in all of the asthmatic patients exposed to pigeons. The MUC1 was upregulated in 15 and downregulated in 2 patients. A higher concentration of MUC1 was seen in younger patients. CONCLUSION: Pigeon droppings and feathers allergen exposed and sensitivity showed the higher MUC1 expression in serum in asthma patients. The concentration of MUC1 was higher in the younger age group. The HPLC method was easier and economical for the detection of MUC1.

How to cite this article:
Mavi AK, Spalgais S, Kumar M, Singh K, Priya, Kumar R. Qualitative analysis of protein antigen in bird exposed asthma patients by high-performance liquid chromatography method.Indian J Allergy Asthma Immunol 2021;35:55-60

How to cite this URL:
Mavi AK, Spalgais S, Kumar M, Singh K, Priya, Kumar R. Qualitative analysis of protein antigen in bird exposed asthma patients by high-performance liquid chromatography method. Indian J Allergy Asthma Immunol [serial online] 2021 [cited 2022 Aug 8 ];35:55-60
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Asthma is a chronic airway inflammatory disease that is complex and it is a result of a combination of genetic predisposition with exposure to inherited allergens. This may provoke allergic reaction and irritate the airway leading to bronchial hyperresponsiveness, underlying inflammation, bronchial airway constriction, and variable recurrent symptoms. The common symptoms of asthma are coughing, shortness of breath and wheezing. It has been shown that asthma is linked with enhanced sensitivity of the tracheobronchial tree in response to a range of stimuli, either separately or in combination with one another. Increased expiratory resistance manifests itself in a variety of ways, including bronchospasm, airway inflammation, mucosal edema, and mucus plugging.[1]

Pigeons are ubiquitous city and household birds found in nearly every neighborhood. Pigeon fancying is one of the humankind's oldest traditions, and it is presently a well-established profession in many parts of the world, as well as a popular recreational activity. It has long been recognized that keeping pigeons as pets or for a living can lead to allergic rhinitis, asthma, and pigeon breeder's lung. According to many immunological investigations, pigeon exposure might cause increasing inflammation in the airway of asthmatic patients. It has been recognized that the exposure to pigeon allergens is associated with various airway diseases, including asthma, rhinitis, and pigeon breeders' disease.[2],[3],[4] The immune complex-mediated hypersensitivity to pigeon is well-known phenomenon. However, Type-I hypersensitivity due to pigeon allergen is limited. It has been also shown that nearly 15% of asthmatics showed Type I hypersensitivity reaction to pigeon allergen. These allergens come from various parts of the pigeon commonly seen in feathers and droppings. The subgroup of asthma patient with a history of pigeon exposure might be the responsible for recurrent symptoms and exacerbation of disease.[2],[5]

Pigeon allergens can be present in the environments without domestic pigeon exposure. It was also shown in a study that an extensive amount of pigeon allergens was exists in school and other environments without breeding of pigeons at these places. They found that the most elevated level of pigeon allergen inhibitory activity was seen in pigeon coop bedding sample with median activity of 11.2% relative to pigeon droppings. The pigeon allergen inhibitory level was generally low in the home and hospital samples, but >50% of samples were still having above detection limit.[4] Various respiratory infectious pathogens such as aspergillus fumigatus, cryptococcus neoformans, chlamydia, mycoplasma, influenza, and Histoplasma may also spread with pigeon droppings. The exposed to pigeon allergens not only increases the hypersensitivity pneumonitis but also have a risk of infections with above-mentioned pathogens.[6]

Human MUC1 (also known as Mucin1, Cell Surface Associated [MUC1]) is a mucin protein that is encoded by the MUC1 gene and found on the surface of various cells. It is also referred to as polymorphic epithelial mucin, epithelial membrane antigen (EMA), or EMA.[7] MUC1 is a glycoprotein with an extracellular domain that is highly glycosylated on the O-linked glycosylation chromosome.[8] Mucins are present on the external surface of epithelial cells of the various organs such as lungs, stomach, intestines, eyes, and other organs, and they help in protection the cells from infection.[9] The MUC1 has been investigated as marker for diagnosis/prognostic of various respiratory diseases including sarcoidosis, asthma hypersensitivity pneumonia (HP), and idiopathic pulmonary fibrosis.[10],[11]

There are various methods for measurement of MUC1 in human serum sample. The high-performance liquid chromatography (HPLC) method is easier and more economical for detection than the Western blot method. We can also do the purification and quantification of antigens with this method. The goal of this study is to examine the expression of serum MUC1 in pigeon exposure asthmatics by HPLC technique.

 Materials and Methods

It was a prospective observational study done at one of the tertiary Chest Institute of India from June 2018 to June 2020. The study enrolled 108 diagnosed cases of bronchial asthma (diagnosed as per GINA guideline) with past of exposure to pigeon's antigens in the past 2 years. The exposed to pigeon allergens was considered positive when a patient of asthma giving a history of exposure to pigeon antigens at home or workplace for at least of 1–3 h/day for at least 6 months in the last 2 years. Skin prick testing (SPT) with pigeon antigens (droppings and feathers) was performed in all the enrolled patients. Patients' demographic details with clinical history and examination and investigation findings were entered into a standard questionnaire. A blood sample 6 ml was collected from all the SPT-positive patients for the analysis of MUC1 proteins. The MUC1 protein analysis was done HPLC method. We exclude the patients who have co-existing diseases, i.e., COPD, hypertension, heart disease from the present study. The study was conducted after approved by the institutional ethics committee. We have also taken written consent for the study from each participant. The diagrammatic presentation of the study design is shown in [Figure 1].{Figure 1}

Liquid chromatography is a commonly used technique for separating a mixture of samples into their constituent elements. It is universally acknowledged. This separation is reliant on the chemical or the actual relationships of the sample mixture with the mobile and stationary phases throughout the separation process. As a result of the large number of stationary/mobile phase combinations that may be used when separating a mixture, there are a number of different types of chromatography. Liquid-solid chromatography, the most widely used chromatography technique, is characterized by the use of a liquid mobile phase that filters down through a solid stationary phase, carrying the separated components with it.[12]

It depends on the pump to pass a compressed liquid containing the sample mixture to undergo a segment loaded up with a solid and powerful adsorbent. Every particle in the sample associates somewhat diversely with the adsorbent material, causing distinctive transportation rates for the various segments and prompting the partition of the segments as they stream out the segment.[13]

Skin prick test in asthmatic patient

Skin prick test was done in all the enrolled patients. Which included a battery of 58 specific environmental/aeroallergens as well as pigeon antigens, such as droppings and feathers. All Cure Pharma Pvt. Ltd. (New Delhi, India) provided the allergen extract in the form of 50% glycerinated solution (1:10 w/v). Negative and positive controls were employed, respectively, with buffered saline and histamine. SPT was performed by putting a drop of antigen on the healthy skin of the forearm volar surfaces and pinching it with sterile needles of 26.5 gauge. When comparing the skin test reactions to the negative control wheal size, i.e., phosphate buffer saline the mean diameter was calculated at the largest breadth of diameter after 15–20 min. In order to qualify as a positive reaction to an allergen, the mean wheal width must be 3 mm or more and must be bigger than the negative control value (buffered saline). Oral medicines, such as antihistamines and any other medications that might have an influence on SPT, were ceased 1 week before the testing, however, inhalation medications were allowed to be used throughout the tests.[14]

MUC1 analysis using high-performance liquid chromatography

The analysis of MUC1 was done by HPLC method. We were used a standard of MUC1 antibody of Bioss, based in the United States, provided the MUC1 standard. HPLC was done with C-18 octadecylsilane column, 2487 UV detector, and 1 ml/min flow rate.

Mobile phase

The derivatizing reagent was used to derivate the sample and standard in a ratio of ethanol: phenylisothiocyanate: triethylamine: water (7:1:1:1). The lyophilized materials were resuspended in mobile phase A after being derivatized (sodium acetate buffer: acetonitrile; 98:2). Waters HPLC system (USA) with Waters 515 HPLC pump, Waters 717 plus autosampler, and Waters 2487 UV detector was used for HPLC analysis. The gradient flow rate (1 ml/min) of the mobile phase A (20 mM sodium acetate: acetonitrile 100%; 98:2; pH 5.1) and mobile phase B (acetonitrile: water) in a 60:40 ratio was maintained in a symmetric C18 250 mm × 4.7 mm ID; 5 m column (Waters, USA). Peaks were detected using a PDA detector, and spectra were retrieved at a wavelength of 254 nm.

Antibody preparation in derivatizing agent was of the same concentration and ratio. The preparation of antibody solution was done by mixing the component in the following ratio. (Ethanol: PITC: Purified Water: Triethanolamine = 350 μl: 50 μl: 50 μl: 50 μl). Here water contains 12.5 μl of Antibody + 37.50 μl of Water. Total 500 μl solution prepared and adds 500 μl of mobile phase A and Lyophilise at 40°C for 3 h. After the completion of the procedure, the solution was kept at 20°C.

Serum sample preparation in derivatizing agent

The preparation of serum solution was done by the mixing the following components (Ethanol: PITC: Serum: Triethanolamine = 350 μl: 50 μl: 50 μl: 50 μl). Before leading to preparation of serum sample preparation, we centrifuge the blood sample, at 5000 RPM at 4°C and separated 50 μl of supernatant liquid for the addition in serum sample preparation. A total of 500-liter solution was prepared then add 500-L of mobile phase A and lyophilized at 400°C for 3 h. The solution was maintained at 200°C once this operation was completed. The gradient flow rate (1 ml/min) of the mobile phase A (20 mM sodium acetate: acetonitrile 100%; 98:2; pH 5.1) and mobile phase B (20 mM sodium acetate: acetonitrile 100 percent; 98:2; pH 5.1) was maintained in a symmetric C18 250 mm × 4.7 mm ID; 5 mm column (Waters, USA) (acetonitrile: water 60:40). Peaks were detected using a PDA detector, and spectra were retrieved at a wavelength of 254 nm. The gradient of flow rates of both phases with curve and time is shown in [Table 1].{Table 1}


The mean age of the study population was 29.83 ± 9.92 years with 60 (55%) patients were female while the remaining 48 (44.4%) were male. All the patients were diagnosed case of bronchial asthma with a positive history of pigeon exposure. All the patients were on the asthma medication for their disease control. SPT reaction showed positive against one of the pigeon allergens in the 17 patients (i.e., either dropping or feather). All the patients were atopic on SPT with positive for at least one of the common aeroallergens. The total IgE was also raised in all 17 patients with mean of 637.8 ± 194.9 IU/mL.

High-performance liquid chromatography analysis of MUC1 protein

The frequency of MUC1 protein in the serum of pigeon antigens (droppings and feathers) exposed and susceptible patients were analyzed using the HPLC technique. All 17 patients were found positive for MUC1. A standard sample was used as a reference for MUC1 analysis. The degree of MUC1 was higher in all 17 samples concerning the normal patient sample. [Table 2] and [Figure 2] show the details of the MUC1 concentration with protein fold of all the enrolled patients. The mean of MUC1 concentration was (2231.9 ± 1070.7 μg/ml) and of 812.8 μg/ml in normal reference subject without exposure to pigeon allergens. The content of MUC1 in serum samples with retention time, area, and concentration details is shown in [Table 3].{Figure 2}{Table 2}{Table 3}

The highest MUC1 concentration with fold change (5342.5 μg/ml and 6.57-fold) was recorded in patient 3 (P3) followed by P6 (3131.9 μg/ml and 3.85-fold) and P14 (3130.1 μg/ml and 3.85-fold). However, P12 (1066.5 μg/ml and 1.31-fold) and P16 (1075.8 μg/ml and 1.32-fold) showed downregulation of MUC1 protein in [Figure 1] and [Figure 2]. Notably, the high concentration of MUC1 in human serum was found in three patients in the 18–23-year-old age range [Figure 3].{Figure 3}


The purpose of the study is to look at the growing quantity of MUC1 protein that has been discovered in asthmatic patients over the past few years. A (HPLC) study was performed to identify the concentration of MUC1 in the sample. With the use of the (HPLC) technology, we were able to determine the frequency of MUC1 concentration in the blood of persons who were exposed to pigeon antigens (droppings and feathers) and were sensitive to them. Chronic bronchial inflammation is a hallmark of asthma, which is an allergic airway disease caused by TH2 cells and IgE in the airways that manifest itself as acute bronchial inflammation. Inhaled allergens and nonspecific toxins such as smoking, cold air, and perfume make the airways of those who suffer from atopic asthma more vulnerable to inflammation. Animal waste such as pigeon droppings and feathers include allergens that have been shown to trigger allergic reactions in people suffering from respiratory diseases such as hypersensitivity pneumonitis and asthma.[15] The MUC1 expression level was higher in patients with exposed history of pigeon droppings and feathers. The above findings of higher MUC1 in pigeon allergens exposed to asthmatic can conclude that these pigeon allergens (droppings and feathers) may be a responsible trigger for asthma symptoms and exacerbation. Pigeons are popular as both pets and city birds, and they are frequently found in every environment. Pigeon numbers in cities have risen as a result of increasing industrialization and urbanization, and pigeon-human interaction has increased as a result of this increase in population. It is possible to find sensitizing allergens in pigeon droppings and feathers, which can trigger an allergic reaction in certain people.[16] Previously, researchers discovered that pigeon exposure was associated with symptoms such as dyspnea, nonproductive cough, lung opacities, increased levels of antibodies to pigeon droppings, and even mortality in certain cases.[17]

MUC1 is expressed on the various surface of secretory glands and a variety of hematopoietic cells as well as epithelial cells in the upper and lower respiratory airways. In addition to being examined as a marker for various cancer diagnosis and its immunotherapy, MUC1 (KL-6) is also being investigated as marker of different lung diseases including pigeon droppings and feathers exposed to asthma.[18],[19] In the present study, all the 17 cases with exposure history and SPT positive showed MUC1 expression in their blood sample. MUC1 was found to be up-regulated in 15 of the patients (88.2%), whereas it was found to be down-regulated in just 2 of the patients (11.8%). According to previous research, asthma patients who are allergic to pigeon allergens (droppings and feathers) or who have other bird-related hypersensitivity pneumonitis had higher serum KL-6 levels than the general population.[20],[21] Imai T et al., in a study reported that serum KL-6 was elevated in acute exacerbation of bronchial asthma of pediatric patients and suggested that KL-6 may be useful marker for asthma diagnosis and prognosis.[22] Chen et al., in a review of MUC1, it was reported that MUC1 was may also use as a biomarker for pigeon-sensitive asthma patients.[19] Hence, the MUC1 expression in serum can be used as diagnostic or prognostic marker of asthma among pigeon-exposed patients in near future. The expression of MUC1 in blood sample can be easily performed by HPLC method.

And with this method, the expression of MUC1 can also use for purification and quantification of antigens. This method is easier and less economical for MUC1 detection in serum samples than the Western blot method.

The MUC1 expression in blood of asthmatic patients is also affected by various factors associated with asthma and its treatment. Hence, the level of MUC1 should be cautiously used with reference to asthma, its treatment and recent infection. The inhaled corticosteroid is the mainstay treatment of asthma. The corticosteroid anti-inflammatory effects may affect the MUC1 expression as it is mediated by MUC1- computed tomography/glucocorticoid receptor nuclear transcription complexes.[23] Furthermore, various bacterial and viral infection are commonly seen in acute exacerbation of asthma. Hence, the up/downregulation of MUC1 may be affected in severe asthma by recent infection.[24] The downregulation of MUC1 in two cases in the present study might due to long-term steroid or near recent exacerbation. There is a need of further study of MUC1 expression in various asthma stages with control status for confirmation of our finding of higher expression of MUC1 in asthmatic. Hence, the MUC1 level may helpful for the treatment of pigeon-exposed asthmatic. The MUC1 expression may become a marker for asthma diagnosis and treatment responsiveness in the near future. We advised for a larger multi-centric study of MUC1 in asthma with different phenotypes and disease stages.


Recent investigations have shown that pigeon antigens correlate with MUC1 protein expression in individuals with asthma, which makes HPLC testing extremely cost-effective. Notably, the high concentration of MUC1 in human serum was found younger age pigeon-exposed asthma patients. The use of HPLC is beneficial in identifying distinct types of MUC1 protein folds and it also may be serves as a biomarker for asthma diagnosis in near future.

Financial support and sponsorship

Author is thankful to Indian Council of Medical Research (ICMR) New Delhi, India for providing grant to carry out this research.

Conflicts of interest

There are no conflicts of interest.


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